Polymers for reversible photoinduced sol gel transitions

ABSTRACT

The present invention provides crosslinked polymeric networks that are reversibly crosslinked upon exposure to light of a suitable wavelength. In one embodiment photocrosslinkable branched hydrophilic polymers containing photochromic groups are synthesized. Cinnamylidene groups and derivatives of cinnamylidene are preferably used as the photochromic agents or photocrosslinking agents.

FIELD OF THE INVENTION

The present invention relates to photoreversible polymeric networks and,particularly, to polymers capable of undergoing photoinduced sol geltransitions.

BACKGROUND OF THE INVENTION

Polymeric networks formed through the action of UV/visible radiation aregenerally constructed via a free-radical reaction initiated bysmall-molecule photosensitive activators. These networks can be based oneither crosslinkable, preformed polymers or by reactions of monomerswith di- and tri-functional crosslinking agents. Crosslinked polymericnetworks are found in a wide variety of industrial and medical uses.

Recently, there has been significant interest in synthesizingcrosslinked hydrophilic polymers for various biological uses. Hydrogels(that is, crosslinked hydrophilic polymers) generally, because of theircharacteristic properties such as swellability in water, hydrophilicity,biocompatibility and lack of toxicity, have been utilized in a widerange of biological and medical applications. The anti-thrombogenic andbiocompatibility properties of poly(ethylene glycol) (PEG), for example,have been extensively studied and are well established. In the case ofexisting PEG-based networks, the crosslinkable groups are traditionallyacrylate or methacrylate groups, which readily polymerize in thepresence of 365 nm radiation and the appropriate activators. Thecarbon-carbon bonds formed during the cross-linking reaction arerelatively permanent, depolymerizing only at elevated temperatures whichwould likely destroy the entire material. In a number of applications,however, it would be beneficial to develop reversibly cross-linkedhydrogels.

Although conventional chemical crosslinking has been extensively used asa hydrogel preparation method, relatively little work has been reportedon the preparation of hydrogels via photopolymerization of water solublepolymers. Current photoinduced systems for hydrogel preparation include:(i) free radical polymerization initiated by long wave ultraviolet lightor visible light of acrylate groups attached to water soluble polymers,and (ii) photopolymerization of photosensitive groups such as cinnamate,stilbazolium or coumarin which are pendant to the end of hydrophilicpolymers. Although such work has demonstrated photopolymerization, therehas been no demonstration of a truly photoreversible system. Indeed,only very limited photoreversibility has been observed.

In that regard, at least one study has reported a photopolymerizedsystem comprising a water soluble polymer having a cinnamate pendantgroup, but the extent of photoreversibility of that system, as observedby UV spectroscopy, was less than 25% (as measured by the change of theabsorbance at 275 nm). In addition, the time of irradiation that wasrequired to detect such reversible behavior was quite long (that is, onthe order of 2 to 3 hours). Likewise, coumarin groups have been reportedto produce photocrosslinked polymer systems based on polyoxazolines. A55% conversion of the photoinduced dimer to the starting material hasbeen reported for such polyoxazoline-based systems. The conversion wascalculated from the UV absorbance spectrum after irradiation of thepolyoxazoline gel with a low-pressure Hg lamp. The time of irradiationof the polyoxazoline-based systems was quite long, however. Moreover,only the photoreversible behavior of the polyoxazoline-based systemsafter a single cycle of irradiation was investigated.

It is very desirable to develop efficient photoreversible hydrogelsystems. Indeed, it is very desirable to develop efficientphotoreversible polymeric crosslinked systems generally (includinghydrophobic systems).

SUMMARY OF THE INVENTION

Accordingly, the present invention provides crosslinked polymericnetworks that are reversibly crosslinked upon exposure to light of asuitable wavelength. In one embodiment photocrosslinkable "branched,""starburst" or other "tree-like" polymers containing photochromic groupsare synthesized.

Preferably, photochromic groups for use in the present invention haverelatively high quantum efficiency for photocrosslinking. Suchphotochromic groups also preferably exhibit a relatively high degree ofphotoreversibility. Cinnamylidene groups, for example, have similarchemistry to cinnamate groups, but have much higher quantum efficiencyfor photocrosslinking. Moreover, the crosslink between cinnamylidenegroups of the present polymer systems has been found to bephotoreversible in that the crosslink between two such groups forms uponexposure to one wavelength of energy and reverts to the original twogroups upon exposure to a different wavelength of energy. To thecontrary, cinnamate groups exhibit relatively low degree ofphotoreversibility. See Photographic Sci. Eng., 15:60 (1971).

Cinnamylidene groups and derivatives of cinnamylidene (for example, acyano derivative) are thus preferably used as the photochromic agents orphotocrosslinking agents. In general, any photochromic cinnamylidenederivative or moiety can be used, including, but not limited to thefollowing: cinnamylidene acetyl chloride, α-methylcinnamylidene acetylchloride, α,γ-dimethylcinnamylidene acetyl chloride,α-phenylcinnamylidene acetyl chloride, α-phenoxycinnamylidene acetylchloride, and cyanocinnamylidene acetyl chloride.

Generally the present invention provides a photosensitive polymernetwork synthesized from branched macromeric precursors. To create acrosslinked network, the branched macromeric precursors are preferablyfunctionalized with at least three photochromic moieties.Functionalization, with less than three photochromic moieties results ina termination point. As set forth above, these photochromic moieties arepreferably cinnamylidene moieties.

The double bonds of such cinnamylidene moieties undergo intermolecularcross-linking via a 2+2 cycloaddition upon exposure thereof to light ofa known range of wavelengths (that is, wavelengths in excess ofapproximately 300 nm) to from the photosensitive polymer network. Thephotosensitive polymer networks formed in this manner are capable ofundergoing a reversible photoscission upon exposure thereof to light ofa second known range of wavelengths. The photoscission reaction occursupon exposure of the photosensitive polymer network to light having awavelength less than 300 nm. Such light is preferably in the range ofapproximately 244 nm to approximately 264 nm.

Preferably, the branched macromeric precursors used in the presentinvention have a molecular weight of at least approximately 400. Giventhe ease of functionalization of a specific macromeric precursor with asuitable photochromic crosslinking group, virtually any macromericprecursor can be used in synthesizing the present photoreversiblepolymeric networks. By way of example only, such macromeric precursorsinclude poly(ethylene oxides), polyoxylates, polycarbonates,polyurethanes and polyacrylates. The branched macromeric precursors arealso preferably functionalized with multiple photochromic moieties (forexample, cinnamylidene moieties) such that the resultant functionalizedprecursor comprises no more than approximately 10 mol % of thephotochromic moiety. More preferably, the branched macromeric precursorsare functionalized with multiple photochromic moieties such that theresultant functionalized precursor comprises no more than approximately5 mol % of the photochromic moiety. The content of the photochromicmoiety in the functionalized precursor is preferably maintainedrelatively low such that the bulk properties of the precursor are notsignificantly affected by the functionalization. It has been discoveredthat in this manner, photoreversible polymeric networks having a widerange of physiochemical characteristics can be synthesized.

In a preferred embodiment, hydrophilic branched macromeric precursorsare used to synthesize a photoreversible hydrogel. Examples of suchhydrophilic macromeric precursors include, but are not limited to, oneor more of the following: poly(ethylene glycol) (PEG), poly(vinylalcohol) (PVA), poly(propylene glycol) (PPG), and polyurethanes.Preferably the hydrophilic branched macromeric precursors are based onPEG.

As used in connection with the present invention, the termphotoreversible refers generally to the capability to undergo aphotoscission of preferably at least 20% of the photochromicintermolecular crosslinks of the present polymer networks. Morepreferably, at least 40% of the intermolecular crosslinks undergophotoscission. Even more preferably, at least 60% of the intermolecularcross-links undergo photoscission. Most preferably, at least 80% of theintermolecular cross-links undergo photoscission.

Preferably, the desired degree of photoscission occurs relativelyrapidly. In that regard, the desired degree of photoscission ispreferably achieved in less than approximately 2 hours. More preferably,the desired degree of photoscission is achieved in less thanapproximately 1 hour. Most preferably, the desired degree ofphotoscission is achieved in less than approximately 0.5 hour.

The photoreversible behavior hydrogel systems under the presentinvention (in water) is quite superior to known photoreversible systems.Indeed, a minimum of 51% (as determined by UV) photoreversibility of PEGsystems under the present invention has been achieved upon irradiationof the solution polymer with a Xenon lamp (150 W) at 254 nm. Inaddition, the reverse reaction occurs at a much faster rate than that ofknown systems. Moreover, the use of a mercury lamp as a light source forinitiating the reversible reaction of the present photoreversiblehydrogels would improve both the extent and rate of the photoreversiblereaction. In that regard, mercury lamps exhibit much higher lightintensity at 254 nm than the Xenon lamp used in the present studies.

Photoreversible hydrogels under the present invention are suitable for anumber of uses. For example, encapsulation of cells for generation of anartificial organ could be accomplished by slurring the cells with aliquid precursor, followed by a chemical or physical reaction whichgenerates a biocompatible gel. While use of the precursor allows ease ofprocessing, reaction to form a more solid construct provides thescaffolding on which the cells can grow and enough structural integrityto allow implantation. Formation of immunoisolative capsules aroundislets of Langerhans (to ultimately allow xenografts as a treatment fordiabetes), for example, requires an initial liquid solution of precursorto form the capsules which then reacts to form a network. The finalcrosslinked system prevents physical degradation of the capsules afterimplantation.

Design of a "bandage" which promotes wound healing could also benefitfrom such an approach. Ideally, a liquid precursor is used which readilyconforms to the irregular contours of the wound and subsequently reactsto form a rigid construct to remain in place as long as necessary.Indeed, polyethylene glycol has been shown to be one of the morebiocompatible synthetic materials which one can choose to constructdevices for use in contact with living tissue, and thus the presentphotocrosslinkable material could have numerous such applications intissue-contact devices. As used herein, the term "biocompatible" refersgenerally to the capability of coexistence with living tissues oforganisms without causing harm thereto.

Moreover, photo-reversibility allows for several other potentialapplications. For example, disposable diapers employ hydrogels(crosslinked, hydrophilic polymers) as the primary absorbents. Theseproducts cannot be recycled because of several factors, among which isthe fact that once crosslinked, a polymer cannot be reprocessed withoutsevere degradation. Consequently, use of an efficient reversiblecrosslinking agent could allow for design of a recyclable hydrogelmaterial.

In general, any application which calls for a recyclable or removablecrosslinked polymeric material would benefit from the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the crosslinking reaction between four-armed PEG andcinnamylidene acetyl chloride.

FIG. 2 illustrates the photopolymerization and photoscission reactionsof four-armed polyethylene glycol-cinnamylidene acetate (b-PEG-CA) whenirradiated with light of an appropriate wavelength.

FIG. 3A illustrates a ¹ HNMR spectrum of b-PEG-cinnamylidene acetate(69% degree of substitution) in D₂ O.

FIG. 3B illustrates the ¹ HNMR spectrum of b-PEG in D₂ O.

FIG. 4 illustrates the UV absorbance spectrum of b-PEG-cinnamylideneacetate.

FIG. 5 illustrates the UV absorbance spectra of cinnamylidene aceticacid and cinnamylidene acetyl chloride in dichloromethane.

FIG. 6A illustrates the absorption spectrum for b-PEG-CA as a functionof time when exposed to light at 313 nm.

FIG. 6B illustrates two cycles of photopolymerization/photoscissionreactions of b-PEG-cinnamylidene acetate as monitored by a UVspectrophotometer equipped with a 150 W Xenon lamp.

FIG. 7 illustrates a dry b-PEG-CA hydrogel (69% modification).

FIG. 8 illustrates the hydrogel of FIG. 7 after swelling in water for 10minutes.

FIG. 9A illustrates a bar graph of percent platelet deposition on PMMAcoverslips and on PMMA coverslips coated with b-PEG-CA hydrogel.

FIG. 9B illustrates an image of platelet deposition on a PMMA coverslipafter 7 minutes of perfusion.

FIG. 9C illustrates an image of platelet deposition on a PMMA coverslipcoated with b-PEG-CA hydrogel after 7 minutes of perfusion.

FIG. 10 illustrates the fraction of the esterification PEG product thathas zero, one, two, three and four cinnamylidene acetyl groups asdetermined using a binomial distribution.

FIG. 11 illustrates a schematic representation of a UV photolytic systemused in the present studies.

FIG. 12 illustrates a schematic representation of a flow chamber used inthe present studies.

DETAILED DESCRIPTION OF THE INVENTION

In the present studies, polyethylene glycol was used as a modelprecursor in the synthesis of photoreversible polymer networks."Four-armed" polyethylene glycol (b-PEG, MW=15,000) was purchased fromShearwater, Inc. Trans-Cinnamaldehyde (99%+), used for the synthesis ofcinnamylidene acetic acid, was purchased from Aldrich Chemical Companyof Saint Louis, Mo. Tetrahydrofuran (THF) (HPLC Grade), used as asolvent for the esterification reaction between cinnamylidene acetylchloride and b-PEG, was purchased from Aldrich Chemical Company. Allpurchased solvents and reagents were used as purchased with no furtherpurification.

Synthesis of Cinnamylidene Acetic Acid

Cinnamylidene acetic acid was synthesized according to the methoddescribed in U.S. Pat. No. 3,257,664, the disclosure of which isincorporated herein by reference. In general, 104 gms of malonic acid(Aldrich), 126 ml of trans-cinnamaldehyde and 90 ml of pyridine weremixed and exposed to slight heating for complete solubilization of thereaction mixture. Piperidine (0.25 ml) was then added, and the mixturewas allowed to stand at room temperature for approximately 26 hours. Thereaction mixture was then refluxed at 115° C. for 18 hours. When thereaction was completed, the reaction mixture was allowed to cool to roomtemperature. In turn, the solution was poured into cold dilute (5 w/v %)hydrochloric acid. The resulting yellowish solid cake was washedrepeatedly with deionized water, and the wet product was crystallizedfrom ethyl alcohol to give cinnamylidene acetic acid (65.22 gms). Thepurity of cinnamylidene acetic acid was determined by ¹ HNMR and UVspectroscopy. ¹ HNMR (CDCl₃ -TMS): δ=6.1 ppm (d, 2H), δ=6.9 ppm (t, 2H),δ=7.5 ppm (m, 5H). The --COOH proton could not be detected in the NMRspectrum, but the product of the esterification reaction betweencinnamylidene acetic chloride and polyethylene glycol established itsexistence. UV (in dichloromethane): λ_(max) =313 nm, ε=37,390 L³ mol⁻¹cm⁻¹, λ_(max) =250 nm, ε=1985 L³ mol⁻¹ cm⁻¹.

Synthesis of Cinnamylidene Acetyl Chloride

Cinnamylidene acetyl chloride was prepared by stirring approximately17.15 gms of cinnamylidene acetic acid with 30 ml thionyl chloride in300 ml of petroleum ether for 12 hours at room temperature. The reactionmixture was then refluxed for 4 hours in an oil bath at a temperature ofapproximately 40 to 50° C. The excess thionyl chloride and the petroleumether were removed under reduced pressure. ¹ HNMR: δ=6.2 ppm (d, 2H),δ=6.9 ppm (q, 2H), δ=7.5 ppm (m, 5H). UV (in dichloromethane): λ_(max)=333 nm, λ_(min) =256 nm.

Synthesis of the Photosensitive b-PEG-Cinnamylidene Acetate

The photosensitive polymer was prepared by a crosslinking reactionbetween b-PEG (four-armed) and cinnamylidene acetyl chloride asillustrated schematically in FIG. 1. The photoreversible nature of thephotopolymerization reaction is also illustrated schematically in FIG.2. FIGS. 3A and 3B illustrate the ¹ HNMR spectra of b-PEG andcinnamylidene acetate-modified b-PEG (69% degree of substitution) indeuterium oxide (D₂ O). The degree of substitution was 0.6575 mol %content of cinnamylidene acetate (CA) as determined by UV. Theappearance of characteristic proton peaks at δ=7.5 ppm, δ=6.9 ppm andδ=6.1 ppm indicated the presence of the cinnamylidene acetyl moiety as apendant group of the b-PEG molecule. The degree of substitution ofphotosensitive groups can also be calculated from the integral ratiobetween the cinnamylidene acetate and the polyethylene glycol (whichappeared at δ=3.6 ppm). Both the UV method and the ¹ HNMR method ofcalculating the degree of substitution (or modification) of the b-PEGmolecule provided similar results.

The UV spectrum of the b-PEG-cinnamylidene acetate (b-PEG-CA) (degree ofsubstitution determined to be 69% by UV, 1.11×10⁻⁶ moles/L) indichloromethane is shown in FIG. 4. Unmodified b-PEG in dichloromethaneshows no significant absorption from 240 nm to 360 nm. Therefore, thecharacteristic absorption peak of b-PEG-CA at 313 nm indicated thecinnamylidene acetate moiety. The UV absorption spectrum of thephotosensitive b-PEG, resembles the UV spectrum of poly(vinylcinnamylideneacetate) compounds which have been investigated and wellcharacterized. See U.S. Pat. No. 3,767,415; Tanaka, H. et al.,"Photoreversible Reactions of Polymers Containing CinnamylideneDerivatives and the Model Compounds," J. Poly. Sci., 15:2685-2689(1977); Tanaka, H. et al., "Photochemistry of Poly(vinylcinnamylideneacetate) and Related Compounds," J. Poly. Sci.,10:1729-1743 (1972); Tanaka, H. et al., "Photosensitivity ofPolyvinylesters of Substituted Cinnamylideneacetic Acids," J. Poly.Sci., 10:3279-3287 (1972); and Tanaka, H. et al., "PhotoreversibleReactions of Poly(vinyl cinnamylideneacetate) and its Model Compound,"J. Poly. Sci., 12:1125-1130 (1974). The UV absorption spectra ofcinnamylidene acetic acid (CAA) and cinnamylidene acetyl chloride (CAC)are illustrated in FIG. 5. The absorption maximum and minimumwavelengths for CAC (λ_(max) =333 nm, λ_(min) =256 nm) are higher thanthe absorption maximum and minimum wavelengths for CAA (λ_(max) =313 nm,λ_(min) =251 nm). Since the absorption maximum and minimum wavelengthsof the CAA were similar to those for the modified b-PEG, CAA was used asthe model compound for the evaluation of the extinction coefficient ofb-PEG-CA at its absorption maximum (ε=37,390 L³ mol⁻¹ cm⁻¹). The degreeof substitution in b-PEG-CA was then estimated from the UV spectrum ofthe photosensitive polymer by using the absorption extinctioncoefficient of the model compound.

Photogelation of Cinnamylidene Acetate-Modified b-PEG

The photopolymerization of the cinnamylidene acetate polyethyleneglycol, as illustrated in FIG. 1, was performed in a bulk film cast fromdichloromethane solution on a cover slip. A 450 W medium pressure Hglamp at λ>300 nm was used as the UV light source. The reaction wasmonitored by studying the change in absorption at λ_(max) =313 nm of themodified b-PEG dissolved in deionized water. A TV spectrophotometerequipped with a 150 W Xenon lamp was used to measure the absorbancechanges. FIG. 6A illustrates the absorption spectrum for b-PEG-CA as afunction of time when exposed to light at 313 nm. The spectral change isan indirect measurement of the disappearance of the double bond adjacentto the carbonyl group of the cinnamylidene acetate moiety and, at thesame time, the formation of a cyclobutane ring. As discussed above,cinnamylidene acetyl moiety undergoes a 2+2 cycloaddition reaction.Specifically, when trans-cinnamylidene acetyl moiety is exposed tolight, it transforms to a cis-cinnamylidene acetyl group whichsubsequently undergoes photopolymerization via cyclobutane ringformation. As shown in FIG. 6B, 87% of the photoreaction is complete inless than 10 minutes (without use of a photoinitiator), as determined bythe changes in the absorbance of the photosensitive polymer at 313 nm.

Photogelation of photosensitive PEG's cast on glass slides occurred asfast as approximately 10 minutes. The gel point was defined as the pointat which b-PEG-CA monomers formed an insoluble three dimensionalstructure in water upon irradiation with UV light. FIG. 7 illustrates adry b-PEG-CA (69% modification) hydrogel resulting after irradiating for2 hours, washing with deionized water, and drying in vacuum for 6 hours.FIG. 8 illustrates the same hydrogel after swelling in water for 10minutes. Further irradiation gave more rigid gels with reducedswellability. In the presence of erythrosin, photosensitive PEGmolecules cast on cover slips reach gel point in less than 20 minutes.Specifically, gel formation was observed after 15 minutes of irradiation(under the same conditions set forth above) when a solution of b-PEG-CA(0.0084 M) in water was mixed with 2 drops of erythrosin solution (0.056M) in water and cast over a glass slide. The light source was a 450 W Hglamp. No gelation occurred in the absence of the cinnamylidene acetylgroup within the polymer chain (that is, in the case of a non-modifiedb-PEG). A mixture of equal amounts of cinnamylidene acetic acid andunmodified b-PEG was suspended in deionized water and irradiated withthe 450 W UV lamp for more than 3 hours, but no gelation was observed.These results indicate that the intermolecular crosslinking by 2+2cycloaddition reaction of the double bonds of the cinnamylidene acetatemoieties in the modified b-PEG caused the formation of stable gels.

Swelling Properties in Water

The degree of swelling in water is a function of the degree ofmodification of the PEG molecule and the duration of UV exposure. As thedegree of substitution of the hydroxel groups of the PEG molecule (withthe photosensitive group) increased, the resulting gels became morerigid, and the amount of water such gels absorbed decreased. Preferably,a degree of swelling in the range approximately 2 to 100 is obtained ina hydrogel system.

To study the effect of the degree of modification upon swellingproperties, samples of photosensitive b-PEG with a variant degree ofmodification were cast on glass slips from a dichloromethane solution.The polymer films were irradiated with a 450 W medium pressure Hg lampfor 1 hour and their degree of swelling was determined. Afterirradiation, the polymer films were washed with deionized water toremove any unreacted polymer and then dried at room temperature, underair for 2 days. The dried gels were weighed and then soaked in deionizedwater for 24 hours. The swollen gels were removed from solution, driedof any excess of fluid with a tissue, and weighed. The degree ofswelling was determined using the equation (Weight_(wet)-Weight_(dry))/Weight_(dry). In this equation, Weight_(wet) refers tothe weight of the swollen gel, while Weight_(dry) refers to the weightof the dried gel. Table 1 shows the change in the degree of swelling ofprepared photosensitive polymers as the degree of modification isaltered.

                  TABLE 1                                                         ______________________________________                                                   Degree of                                                                     Subs.                                                                               (avg.) of       Weight of                                            Mol %    PEG's    Weight of                                                                            Swollen                                      Number of                                                                             Content  Hydroxyl Dry Gel                                                                              Gel    Degree of                             Experiments                                                                           of CA%   Groups   (mg)   (mg)   Swelling                              ______________________________________                                        1       0        0        no     N/A    N/A                                                             gelation                                            4       0.6575   25%      .775 ±                                                                            46.25 ±                                                                           58 ± 4                                                       .125   9.95                                         4       1.68     64%      3.425 ±                                                                           121.05 ±                                                                          35 ± 5                                                       1.47   39.8                                         ______________________________________                                    

As set forth above, as the time of irradiation increased, the resultantgels became more rigid and their retention of water decreased. In Table2 below, the degree of swelling of the PEG gels (69% degree ofsubstitution) is shown as a function of irradiation time for a 5-hourperiod. Although the degree of swelling decreases with increasingduration of irradiation, even after 5 hours of irradiation, the PEG gelscan still retain a large amount of water (that is, a water uptake of 13times the gel's original weight). The degrees of swelling that have beenreported in this study, appear to be much higher than those of gels thathave been synthesized by photopolymerization of water soluble polymershaving different photocrosslinkable moieties as pendant groups, such ascoumarin and cinnamate groups, under the same irradiation conditions.

                  TABLE 2                                                         ______________________________________                                        Time          Degree of                                                                              Number of                                              (hours)       Swelling Experiments                                            ______________________________________                                        1             >16      1                                                      2             16 ± 1                                                                              3                                                      3             14 ± 1                                                                              3                                                      4             13 ± 1                                                                              3                                                      ______________________________________                                    

Photoreversible reaction

Prior to the present invention, merely a few photoinduced hydrogelsystems have exhibited only a very limited degree of reversibility. Thephotoreversibility of b-PEG-CA was monitored by studying the change ofits UV absorption (69% degree of functionalization) in water at 313 nm.FIG. 6B illustrates two full cycles of the photoreversibility thatb-PEG-CA in water exhibits when exposed to UV light of a properwavelength. For the first 10 minutes of irradiation (the forward,photopolymerization reaction), the absorbance peak at 313 nm decreasedfrom 2.375 to 0.3 (an 87% decrease). At the end of the 10th minute, the300-400 nm bandwidth filter was replaced by a 254±10 nm filter and thereverse, photoscission reaction was observed during the next 4 minutes.During that time, the absorbance peak at 313 nm increased from 0.3 to1.35 (a 51% increase). Subsequently, the 254 nm filter was replaced withthe 300-400 nm filter and the forward reaction was once again studied.The absorbance peak at 313 nm decreased from 1.35 to 0.3 (a 77.7%decrease) during the next 10 minutes of light exposure. Finally, the 254nm bandwidth filter was once again placed in front of the light sourceand the photoreversible reaction was studied. The absorbance at 313 nmincreased from 0.3 to 0.9 in the final 4 minutes of irradiation (a 29%increase).

During the first cycle of photopolymerization-photoscission reaction, a"reversibility" of 51% was observed for the polymer system as determinedby the changes in absorbance at λ=313 nm. In the second cycle of thephotoreaction, a recovery of nearly 30% of absorbance was observed. Theextent of photoreversibility can, however, only be estimated since amixture of cis and trans photocleaved b-PEG-CA molecules are likely tohave been produced from the irradiation at 254 nm. Because theextinction coefficient of the cis-b-PEG-CA molecules is less than theextinction coefficient of the corresponding trans molecules, a lowerabsorbance was observed and, consequently, a smaller extent ofphotoreversibility of the b-PEG-CA hydrogels was calculated. In otherwords, the present method for calculating the extent ofphotoreversibility inherently underestimates the extent ofphotoreversibility and thus provides a minimum value. Visualobservations of the "de-geletion" of the present hydrogels confirmedsubstantial photoreversibility.

The photoscission of the photopolymerized b-PEG-CA molecules in waterappears to be a much faster reaction than the forwardphotopolymerization reaction. The highest absorbance change during thephotoscission reaction is achieved within the first 4 minutes of lightexposure at 254 nm. After 4 minutes of irradiation, only a slightdecrease of the absorbance of the b-PEG-CA is observed. This observancemay be a result of either: (i) some photodecomposition at the higherenergy, shorter wavelength light, or (ii) leakage of the 254 nmbandwidth filter, which would result in some photopolymerization effecton uncrosslinked b-PEG-CA molecules.

The effect of a photosensitizer (Erythrosin) on the photoreaction wasalso investigated. Erythrosin is a photosensitizer which absorbsstrongly between 400 and 500 nm. When b-PEG-CA solution was mixed witherythrosin and irradiated with a 150 W Xenon lamp, the mixture followedthe same photochemical behavior at 313 nm as found in the absence of thephotosensitizer. It is believed that in the presence of appropriatephotosensitizers, hydrogels under the present invention can bephotosynthesized via irradiation with visible light.

Biocompatibility of the b-PEG-CA Hydrogels

The results of blood perfusion studies are illustrated in FIGS. 9Athrough 9C. In FIG. 9A, the data are expressed as percent of surfacearea covered by platelets. In these studies, two polymethylmethacrylate(PMMA) slides and two b-PEG-CA coated PMMA slides were perfused andquantified. As illustrated in FIG. 9A, there was a highly significant(p<0.001) difference between the b-PEG-CA treated slides and the PMMAslides. This difference is illustrated graphically in FIGS. 9B and 9C.The b-PEG CA coating was thus found to be very effective in preventingplatelet deposition. This result indicates that the photocrosslinking ofthe branched PEG under the present invention preserves the known bloodbiocompatibility properties of the polyethylene glycol moleculeprecursor.

Hydrogel Stability

Hydrogels prepared from 1.68 mol % CA content and 0.6575 mol % CAcontent, respectively, were stored at room temperature over a 2 monthperiod. All of the samples retained their hydrophilic nature and waterswellability, indicating that the intermolecular cross-linking by a 2+2cycloaddition reaction of the cinnamylidene acetyl groups were strongenough to maintain the hydrogel structures intact for long periods oftime.

Entrapment of Fluorescein Isothiocyanate Dextran in PEG-CA Hydrogel andSubsequent "De-gelation"

The use of a hydrogel synthesized under the present invention toencapsulate a Dextran was also studied. b-PEG-CA hydrogel fixed withDextran-FITC was subjected to photoscission by irradiation with UV lightfrom a 45 W Hg lamp at 254 nm. The PEG gel before irradiation appearedquite stable, with a light reddish color resulting from the Dextran'sdye. The degree of swelling of the hydrogel was determined to beapproximately 20. The hydrogel was then subjected to UV light at 254 nmfor 20 minutes. The "de-gelation" of the PEG hydrogel was measured bydetermining the release of Dextran into a fixed volume of deionizedwater via fluorescence spectroscopy. Table 3 sets forth the intensity offluorescence at 520 nm of the water solution resulting from the releaseof Dextran out of the hydrogel matrix as a function of irradiation time.As the irradiation time at 254 nm was increased the intensity offluorescence in the water solution increased, indicating that Dextranwas diffusing out of the hydrogel at a faster rate because of reducedcrosslink density caused by photoirradiation at 254 nm. The resultsafter 20 minutes indicate approximately 80% reversion. After 30 minutesof exposure at 254 nm, the hydrogel lost most of its integrity andappeared loose and colorless.

                  TABLE 3                                                         ______________________________________                                        Time        Intensity of Fluorescence at                                      (minutes)   520 nm                                                            ______________________________________                                         0          160.34                                                            10          310.78                                                            20          558.30                                                            ______________________________________                                    

The following examples detail the experimental conditions used in anumber of typical studies under the present invention.

EXAMPLE 1

Synthesis of Branched PEG-Cinnamylidene Acetate

A typical procedure for the synthesis of photosensitive PEG is describedbelow. Typically, 1.5 gms of b-PEG (four-armed, MW=15,000) were placedin a 250 ml, three-necked reaction vial and were dissolved in 80 ml ofTHF (HPLC grade). Some heating was required (T=40 to 50° C.) forcomplete solubilization of the b-PEG in the solvent. When thetemperature of the reaction mixture returned to room temperature, thereactor was purged with 99+% N₂ gas for approximately fifteen minutes.Approximately 1.65 gms of cinnamylidene acetyl chloride which hadpreviously been dissolved in 10 ml of THF were then transferred dropwisethrough a septum into the reaction vial. The reaction mixture wasstirred continuously in the dark for approximately 17 hours at roomtemperature. Finally, the mixture was refluxed for 6 hours at 45 to 50°C. The resulting product was isolated by first removing the solvent byvacuum distillation, then precipitating the product three times indiethyl ether and finally, washing the product three times with acetoneto remove any unreacted cinnamylidene acetyl chloride (CAC).Approximately, 1.05 gms of the modified PEG were collected (70% yield)and the product was placed in a vacuum oven to dry overnight at roomtemperature. ¹ HNMR (CDCL₃ -TMS): δ=3.5 ppm (t, 1362H), δ=6.1 ppm (d,8H), δ=6.9 ppm (q, 8H), δ=7.5 ppm (m, 20H). UV (in dichloromethane):λ_(max) =313 nm (ε=37,390 L³ mol⁻¹ cm⁻¹), λ_(min) =254 nm.

EXAMPLE 2

Photocrosslinking

In a typical experiment, cinnamylidene acetate modified PEG wasdissolved in dichloromethane and then cast over a glass slide at roomtemperature under air. The resulting photocrosslinkable modified PEG wasirradiated with a 450 watt medium pressure UV lamp (Hanovia). The lampwas placed in a water-jacket well, approximately 15 cm above the coatedslide, and the polymer film was irradiated over a period in the range ofapproximately 15 minutes to 3 hours. A Pyrex filter was used toeliminate the wavelengths below 300 nm. After irradiation was completed,the film was washed with deionized water to remove any unreacted polymerand then was dried in vacuo. The yield of gelation was dependent on thetime of irradiation.

EXAMPLE 3

Determination of Swelling Properties

In a typical experiment, the equilibrium swelling properties of thephotocrosslinkable gel in deionized water were measured by dissolving72.5 mg of b-PEG-CA in 0.4 mL of deionized water from which a film wascast in a cover slip as described above. After 2.5 hours of irradiationwith a 450 W UV lamp, the resulting polymer was carefully dried andweighed. The resulting gel was immersed in 50 mL of deionized water for6 hours at room temperature. The weight of the wet gel was determined tobe 644.6 mg after excess water was carefully removed with a tissue. TheDegree of Swelling was calculated as follows: (Weight_(wet)-Weight_(dry))/Weight_(dry).

EXAMPLE 4

Photoreversibility of the Photosensitive PEG

(a) The photoreversibility of the PEG-cinnamylidene acetate crosslinkingwas monitored using a UV spectrophotometer equipped with a 150 W Xenonlamp. A diagram of the apparatus is illustrated in FIG. 11. A small dropof a 22.5 w/v % solution of b-PEG-CA in deionized water was placedbetween two quartz plates (0.1 mm cell thickness), and thephotopolymerization reaction was monitored for 10 minutes in 2-minuteincrements using a bandwidth filter to eliminate wavelengths below 300nm. At the 10th minute, the filter was replaced with a 254 nm bandwidthfilter (Andover) and the photocleavage reaction was monitored for thefollowing 6 minutes. At the end of the sixth minute, the original300-400 nm bandwidth filter was placed in front of the UV source and theforward reaction (photopolymerization) was observed once again for thenext 10 minutes. Finally, the 254 nm bandwidth filter was placed infront of the UV light source and the reverse reaction was studied for 4minutes. Photoinduced changes in the UV absorption spectrum ofb-PEG-CA's UV were measured between 240 nm to 360 nm.

(b) Fluorescein-Isothiocyanate Dextran (Sigma) (0.04 h, MW=145,000) wasmixed with a 1 mL solution of b-PEG-CA (0.157 g) in water, and thesolution poured into a pyrex dish. The polymer solution was irradiatedwith a 450 W Hg lamp for approximately 2.5 hours. The resultingphotocrosslinked polymer was washed with deionized water to remove anyuncrosslinked polymer and air-dried for two days. The dry gel wasallowed to equilibrate in 40 ml of water for 26 hours. A small piece ofthe purified gel was irradiated with a 450 W lamp at 254 nm inincrements of 10 minutes. Following each irradiation, the gel was placedin 10 ml of fresh deionized water and allowed to equilibrate for 30minutes. The water solutions after each irradiation were collected, andthe Dextran released from the gel matrix was measured by fluorescencespectroscopy at λ=520 nm. A Perkin Elmer Luminescence Spectrometer LS50Bequipped with a Xenon lamp was used for the fluorescence measurements.

EXAMPLE 5

Calculation of Degree of Substitution

(a) The mol % of substitution was calculated as follows. The generalformula for PEG is

    HO--(CH.sub.2 CH.sub.2).sub.x --OH

Given a molecular weight of 15,000 for the PEG macromolecular precursor,there are approximately 340.5 such repeat units per macromolecule. Asthere are four hydrogen protons per repeat unit, there are approximately1362 such protons in a macromolecular precursor having a molecularweight of 15,000. The number of hydrogen protons per attachedcinnamylidene acetate moiety is 9. For 100% attachment of cinnamylideneacetate moiety, the total number of hydrogen protons corresponding tothe cinnamylidene acetate moiety is 36. The degree of modification for100% attachment is calculated via the following equation: ##EQU1##

(b) Using the calibration curve for cinnamylidene acetic acid indichloromethane at 313 nm, the degree of substitution of PEG was alsodetermined. According to the Baer-Lambert Law absorbance (A) iscalculated according to the following formula: A=ε·b·c, wherein ε is theextinction coefficient, b is the cell thickness (1 cm in the presentcase), and c is the concentration. The extinction coefficient determinedfor the model compound, cinnamylidene acetic acid, was 37,390 L³ cm⁻¹mol⁻¹. By dissolving a sample in dichloromethane (at a knownconcentration), one can determine the degree of substitution bymeasuring the absorbance. The relevant equation becomes A=-2.6378×10⁻³+37,390·c. The correlation coefficient (R²) was calculated to be 0.995.

For example, 1.11×10⁻⁶ mol L⁻¹ of b-PEG-CA, exhibited an absorbance of0.1123 at 313 nm. For 100% attachment of CA the concentration of CAwould be 4.44×10⁻⁶ mol L⁻¹. Using the previous equation to solve for theabsorbance corresponding to 100% attachment, results in A=0.16337.Therefore, a measured absorbance of 0.1123 corresponds to a degree ofsubstitution of 68.7% according to the formula 0.1123/0.16337·100%.

Assuming the reactivity of each arm is independent of the status of theother arm, a binomial distribution can be used to estimate thefunctionality of the esterification product. For example, for a PEGmolecule with four arms and an average degree of esterification of 70%(as measured by UV), the resulting product is calculated to comprise0.8% PEG with no cinnamylidene acetyl group attached, 7.6% with onegroup attached, 26.5% with two groups attached, 41.2% with three groupsattached, and 24% with four groups attached (see FIG. 10).

EXAMPLE 6

Biocompatibility of b-PEG-CA Hydrogels.

The anti-thrombogenic behavior of the b-PEG-CA hydrogels was determinedby studying platelet deposition on hydrogel coated glass coverslipsperfused with mepacrine-labeled blood. Platelet deposition was measuredby using Epifluoresent Video Microscopy. All cover slips were cleanedand spin cast with PMMA. Half of the PMMA slides were spin cast withb-PEG-CA monomers and photopolymerized with a 450 W Hg lamp forapproximately 75 minutes. The PMMA slides that had not been coated withb-PEG-CA hydrogels were exposed to UV light for approximately 2 hoursand 15 minutes. The uncoated slides served as a control.

Blood was drawn using a 19 gauge needle from the antecubital vein into 4U/ml Heparin anticoagulant from a healthy nonmedicated volunteer.Heparinized blood was fluorescently labeled with mepacrine. Coverslipswere loaded into a flow chamber (see FIG. 12) and the system was filledwith Phosphate Buffered Saline. Blood was withdrawn through tubing intothe chamber at a wall shear rate of approximately 300 s⁻¹. The chamberwas placed on a Zeiss Axiovert inverted microscope stage and images wererecorded after 7 minutes of perfusion.

Images from the scope were passed through an image intensifier(Hamamatsu) and digitized with a CCD camera (Hamamatsu). Output from thecamera was sent to a computer and recorded on a Super VHS videocasseterecorder for future image analysis. The image acquisition system hasbeen described in Hubbell, J. A. and McIntire, L. V., Rev. Sci.Instrum., 57, 892-897 (1986). During perfusions, images were gathered bymoving the stage to a random spot on the slide and recordingapproximately 5 seconds of flow to the tape. Fifteen separate randomimages were obtained for each slide tested. At a later time, recordedperfusions were played back and images were gathered on a computer usinga BDS imaging software. Each image collected was an integration of 24consecutive frames to cancel the effect of platelets moving in thebackground. For each set of images, the threshold of pixel intensitycorresponding to platelet deposition was determined. Images wereconverted to bitmaps by assigning any pixel above a predeterminedthreshold a value of 1. The percent surface coverage by platelets wascalculated from bitmaps and averaged.

Although the present invention has been described in detail inconnection with the above disclosure and examples, it is to beunderstood that such detail is solely for that purpose and thatvariations can be made by those skilled in the art without departingfrom the spirit of the invention except as it may be limited by thefollowing claims.

What is claimed is:
 1. A hydrophilic, photosensitive polymer networksynthesized from hydrophilic branched macromeric precursors comprisingpolyethylene glycol, the branches of the branched macromeric precursorsbeing functionalized with a cinnamylidene moiety, the double bonds ofthe cinnamylidene moieties undergoing intermolecular crosslinking via a2+2 cycloaddition upon exposure thereof to light of a first known rangeof wavelengths to form the hydrophilic, photosensitive polymer network,the hydrophilic, photosensitive polymer network being suitable toundergo a reversible photoscission upon exposure thereof to light of asecond known range of wavelength.
 2. The hydrophilic, photosensitivepolymer network of claim 1 wherein each of the branched macromericprecursors comprises no more than 5% cinnamylidene moiety.
 3. Thehydrophilic, photosensitive polymer network of claim 1 wherein thecinnamylidene moiety is selected from the group consisting ofcinnamylidene acetate, α-methylcinnamylidene acetate,α,γ-dimethylcinnamylidene acetate, α-phenylcinnamylidene acetate,α-phenoxycinnamylidene acetate, and cyanocinnamylidene acetate.
 4. Thehydrophilic, photosensitive polymer network of claim 1 wherein thebranched macromeric precursors have a molecular weight of at least 400.5. The hydrophilic, photosensitive polymer network of claim 1 whereinthe branched macromeric precursors comprise biocompatible macromers andthe photosensitive polymer network is biocompatible.
 6. The hydrophilic,photosensitive polymer network of claim 1 wherein the branchedmacromeric precursors are functionalized with a cinnamylidene moiety atthe terminus of at least three branches thereof.
 7. The hydrophilic,photosensitive polymer network of claim 6 wherein the cinnamylidenemoiety is selected from the group consisting of cinnamylidene acetate,α-methylcinnamylidene acetate, α,γ-dimethylcinnamylidene acetate,α-phenylcinnamylidene acetate, α-phenoxycinnamylidene acetate, andcyanocinnamylidene acetate.
 8. The hydrophilic, photosensitive polymernetwork of claim 6 wherein the branched macromeric precursors have amolecular weight of at least
 400. 9. A method of synthesizing ahydrophilic, photosensitive polymer network comprising the step ofcrosslinking hydrophilic branched macromeric precursors comprisingpolyethylene glycol, the branches of the branched macromeric precursorsfunctionalized with a cinnamylidene moiety, the double bonds of thecinnamylidene moieties undergoing intermolecular crosslinking via a 2+2cycloaddition upon exposure thereof to light of a first known range ofwavelengths to form the hydrophilic, photosensitive polymer network, thehydrophilic, photosensitive polymer network being suitable to undergo areversible photoscission upon exposure thereof to light of a secondknown range of wavelength.
 10. The method of claim 9 wherein each of thebranched macromeric precursors comprises no more than 5% cinnamylidenemoiety.
 11. The method of claim 9 wherein the cinnamylidene moiety isselected from the group consisting of cinnamylidene acetate,α-methylcinnamylidene acetate, α,γ-dimethylcinnamylidene acetate,α-phenylcinnamylidene acetate, α-phenoxycinnamylidene acetate, andcyanocinnamylidene acetate.
 12. The method of claim 9 wherein thebranched macromeric precursors have a molecular weight of at least 400.13. The method of claim 9 wherein the branched macromeric precursorscomprise biocompatible macromers and the photosensitive polymer networkis biocompatible.
 14. The method of claim 9 wherein the branchedmacromeric precursors are functionalized with a cinnamylidene moiety atthe terminus of at least three branches thereof.
 15. The hydrophilic,photosensitive polymer network of claim 14 wherein the cinnamylidenemoiety is selected from the group consisting of cinnamylidene acetate,α-methylcinnamylidene acetate, α,γ-dimethylcinnamylidene acetate,α-phenylcinnamylidene acetate, α-phenoxycinnamylidene acetate, andcyanocinnamylidene acetate.
 16. The hydrophilic, photosensitive polymernetwork of claim 14 wherein the branched macromeric precursors have amolecular weight of at least 400.